Methods and compositions for treating celiac disease

ABSTRACT

The invention features the treatment of gastrointestinal disorders associated with an innate immune response tiggered by alpha amylase inhibitor CM3, alpha amylase inhibitor 0.19 (0.19), CM1, CM2, CMa, CMd, CM16, CMb, CMX1/CMX3, CMX2, and/or alpha amylase inhibitor 0.53 (0.53). To this end, the invention features pharmaceutical compositions including neutralizing antibodies to CM3, 0.19, CM1, CM2, CMa, CMd, CM16, CMb, CMX1/CMX3, CMX2, and/or 0.53, food products containing reduced levels of CM3, 0.19, CM1, CM2, CMa, CMd, CM16, CMb, CMX1/CMX3, CMX2, and/or 0.53 protein, the use of oral TLR4 inhibitors to block the effect of said alpha-amylase inhibitors, assays for identifying CM3, 0.19, CM1, CM2, CMa, CMd, CM16, CMb, CMX1/CMX3, CMX2, and/or 0.53 content in food products, and assays for diagnosing subjects with a disorder related to CM3, 0.19, CM1, CM2, CMa, CMd, CM16, CMb, CMX1/CMX3, CMX2, and/or 0.53 triggered innate immune responses.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application Nos.61/330,043, filed Apr. 30, 2010 and 61/417,613 filed Nov. 29, 2010, eachof which is hereby incorporated by reference.

STATEMENT AS TO FEDERALLY FUNDED RESEARCH

This work was supported by grant number NIH 1R21AI078385A1-01 from theUnited States National Institutes of Health. The Government has certainrights in the invention.

BACKGROUND OF THE INVENTION

This invention relates to the treatment of celiac disease with compoundsthat decrease inflammation resulting from contact with wheat alphaAmylase inhibitors, including CM3 or 0.19.

Celiac disease (CD) is an inflammatory small intestinal disorder, oftenaccompanied by a malabsorptive syndrome which is caused in part by anuncontrolled immune reaction to ingested gluten proteins. The prevalenceof this disease in affected populations such as the US, the Middle East,or Europe is 0.5-2.5%. Untreated, CD patients can develop serioussecondary morbidity, such as T cell lymphoma and autoimmune diseases.The disorder is considered to be the result of a complex interplay ofintrinsic (genetic) and variable extrinsic (environmental) factors thatexplain the wide spectrum of clinical manifestations ranging fromasymptomatic to severe malabsorption. Gluten peptides are efficientlypresented by celiac disease-specific HLA-DQ2- and HLA-DQ8-positiveantigen-presenting cells, and thus drive the adaptive immune response,predominantly in the connective tissue of the lamina propria. Tissuetransglutaminase (tTG), which has been identified as the highly specificendomysial autoantigen, is increasingly released from cells duringinflammation. It usually potentiates antigen presentation by HLA-DQ2 andHLA-DQ8 by deamidating and cross-linking gluten peptides. The result islamina propria T-cell activation and mucosal transformation by activatedintestinal mononuclear cells and fibroblasts.

Currently, celiac disease is typically treated with a strict gluten freediet. However, a gluten free diet is difficult to maintain andnon-dietary treatment alternatives are urgently needed. Moreover, even agluten free diet may not lead to remission in patients with refractoryceliac disease.

SUMMARY OF THE INVENTION

The invention features a pharmaceutical composition including anantibody (e.g., a polyclonal, monoclonal, or humanized antibody) againstalpha amylase inhibitor CM3, alpha amylase inhibitor 0.19 (0.19), CM1,CM2, CMa, CMd, CM16, CMb, CMX1/CMX3, CMX2, alpha amylase inhibitor 0.53(0.53), and structurally and functionally related molecules(collectively termed AAI). The antibody can be formulated for, e.g.,oral administration (e.g., in milk or colostrum). Furthermore, theantibody can be produced in the milk or colostrum of a mammal (e.g.,goat or cow). Also, the antibody can be formulated to be active in theintestine.

In another aspect, the invention features a method of treating agastrointestinal disorder (e.g., celiac disease, ulcerative colitis,Crohn's disease, or irritable bowel syndrome) by administering any ofthe pharmaceutical compositions described above or an oral or systemicTLR4 inhibitor (e.g., as described below). The pharmaceuticalcomposition can be administered immediately prior to, during or after ameal, or can be administered, e.g., once, twice, or three times daily.

In another aspect, the invention features a method of determining asubstance's potency in inducing a negative gastrointestinal reaction bymeasuring the AAI content of the substance, where the measurement isindicative of the potency of the substance in inducing a negativegastrointestinal reaction. This method can include dispersing all or afraction of the substance in an aqueous solution, contacting thesolution with an antibody specific for an AAI (e.g., an antibody boundto a substrate) under conditions conducive to specific binding, andmeasuring the amount of the antibody bound to the AAI. In oneembodiment, this method features an ELISA assay.

In another aspect, the invention features a method of testing thesensitivity of a subject to ingestion of AAI containing substances bymeasuring the levels (e.g., with an ELISA assay) of anti-AAI antibody insample isolated from the subject (e.g., a blood or stool sample) wherethe levels are indicative of the sensitivity of the subject to ingestionof AAI containing substances.

In yet another aspect, the invention features a method of reducing thepotency of a substance in inducing a negative gastrointestinal reactionby reducing the AAI content of the substance (e.g., through enzymaticdegradation, disulfide reduction, or separation of AAI).

In a related aspect, the invention features a cereal product (e.g.,wheat, rye, barley, oats, corn or rice) including reduced levels of AAIprotein (e.g., through enzymatic degradation, disulfide reduction,separation, or by derivation from cereals engineered to express AAI atdecreased levels) compared to levels of AAI in naturally occurringcereal products.

In another aspect, the invention features a nucleic acid constructencoding an RNAi molecule against AAI and a vector including a nucleicacid construct encoding an RNAi molecule against AAI. The invention alsofeatures a transgenic plant including any of the above nucleic acidconstructs or vectors. These transgenic plants can be processed intofood products with decreased AAI content thereby resulting in lesspotent induction of a negative gastrointestinal reaction in a subjectwith a gastrointestinal disorder (e.g., celiac disease, ulcerativecolitis, and Crohn's disease).

By “wheat alpha Amylase inhibitor AAI”, and specifically “CM3” and“0.19,” is meant any polypeptide having the activity of full-length CM3or 0.19 protein:

CM3: (SEQ ID NO: 1) MACKSSCSLLLLAAVLLSVLAAASASGSCVPGVAFRTNLLPHCRDYVLQQTCGTFTPGSLPEWMTSASIYSPGKPYLAKLYCCQELAEISQQCRCEALRYFIALPVPSQPVDPRSGNVGESGLIDLPGCPREMQWDFVRLLVAPGQCNLATIHNVRYCPAVEQPLWIDYKDDDDK. 0.19: (SEQ ID NO: 2)SGPWMCYPGQAFQVPALPACRPLLRLQCNGSQVPEAVLRDCCQQLAHISEWCRCGALYSMLDSMYKEHGAQEGQAGTGAFPRCRREVVKLTAASITAVCRLPIVVDASGDGAYVCKDVAAYPD

The terms “wheat alpha Amylase inhibitor CM3”, or “CM3”, “wheat alphaAmylase inhibitor 0.19,” or “0.19” also are used herein to refer to CM3or 0.19 fragments, which may be, e.g., functional, antigenic, and/orimmunogenic. Further, these terms also encompass CM3 or 0.19polypeptides or fragments including additional terminal amino acids,e.g., an amino terminal methionine.

By “the activity of full-length CM3 protein”, or “the activity offull-length 0.19 protein” is meant binding to TLR4 and induction of IL-8secretion in monocytes, macrophages, or dendridic cells as describedherein.

The terms “wheat alpha Amylase inhibitor CM3,” “CM3,” “wheat alphaAmylase inhibitor 0.19,” or “0.19” are also used herein to refer toproteins or peptides having at least 20%, e.g., at least 25%, 30%, 40%,50%, 60%, 70%, 75%, 80%, or 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99%) amino acid sequence identity to the sequence of SEQ IDNO:1 or SEQ ID NO:2. Proteins having such amino acid sequence identitycan, e.g., have cysteine residues at positions corresponding to theamino acid residue located in the CM3 protein at residue numbers 29, 43,83, 84, 94, 96, 130, and 159.

The terms “CM1,” “CM2,” “CMa,” “CMd,” “CM16,” “CMb,” “CMX1/CMX3,”“CMX2,” and “alpha amylase inhibitor 0.53” (0.53) are used herein torefer to protein fragments, which may be, e.g., functional, antigenic,and/or immunogenic. Further, these terms also encompass CM1, CM2, CMa, 5CMd, CM16, CMb, CMX1/CMX3, CMX2, and 0.53 polypeptides or fragmentsincluding additional terminal amino acids, e.g., an amino terminalmethionine.

The terms “CM1,” “CM2,” “CMa,” “CMd,” “CM16,” “CMb,” “CMX1/CMX3,”“CMX2,” and 0.53 are also used herein to refer to proteins or peptideshaving at least 20%, e.g., at least 25%, 30%, 40%, 50%, 60%, 70%, 75%,80%, or 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) aminoacid sequence identity to the sequence of SEQ ID NOs: 6-11, respectivelyhaving the activity of binding to TLR4 and induction of IL-8 secretionin monocytes, macrophages, or dendridic cells as described herein.Proteins having such amino acid sequence identity can, e.g., havecysteine residues at positions corresponding to the amino acid residuelocated in the CM3 protein at residue numbers 29, 43, 83, 84, 94, 96,130, and 159.

By “polypeptide,” “polypeptide fragment,” or “peptide” is meant a chainof two or more (e.g., 10, 15, 20, 30, 50, 100, or 175, or more) aminoacids, regardless of any post-translational modification (e.g.,glycosylation or phosphorylation), constituting all or part of anaturally or non-naturally occurring polypeptide, fragment, or peptide.By “post-translational modification” is meant any change to apolypeptide or polypeptide fragment made during or after synthesis.Post-translational modifications can be produced naturally (such asduring synthesis within a cell) or generated artificially (such as byrecombinant or chemical means). A “protein” can be made up of one ormore polypeptides.

The term “identity” is used herein to describe the relationship of thesequence of a particular nucleic acid molecule or polypeptide (or afragment thereof) to the sequence of a reference molecule of the sametype (or a fragment thereof). For example, if a nucleic acid or aminoacid molecule has the same nucleotide or amino acid residue at a givenposition, as compared to a reference molecule to which it is aligned,there is said to be “identity” at that position. The level of sequenceidentity of a nucleic acid molecule or a polypeptide to a referencemolecule is typically measured using sequence analysis software with thedefault parameters specified therein, such as the introduction of gapsto achieve an optimal alignment (e.g., Sequence Analysis SoftwarePackage of the Genetics Computer Group, University of WisconsinBiotechnology Center, 1710 University Avenue, Madison, Wis. 53705,BLAST, or PILEUP/PRETTYBOX programs). These software programs matchidentical or similar sequences by assigning degrees of identity tovarious substitutions, deletions, or other modifications.

The sequence of a nucleic acid molecule or polypeptide is said to be“substantially identical” to that of a reference molecule if itexhibits, over its entire length, at least 51%, e.g., at least 55%, 60%,65%, 75%, 85%, 90%, or 95% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99%) identity to the sequence of the reference molecule.For polypeptides, the length of comparison sequences may be, forexample, at least 10, 15, 20, 30, 50, 100, or 175, or more amino acids.

A molecule, e.g., an oligonucleotide probe or primer, a gene or fragmentthereof, a cDNA molecule, a polypeptide, or an antibody, can be said tobe “detectably-labeled” if it is marked in such a way that its presencecan be directly identified in a sample. Methods for detectably labelingmolecules are well known in the art and include, without limitation,radioactive labeling (e.g., with an isotope, such as ³²P or ³⁵S) andnonradioactive labeling (e.g., with a fluorescent label, such asfluorescein).

By a “substantially pure” polypeptide (e.g., antibody) is meant apolypeptide (or a fragment thereof) that has been separated fromproteins and organic molecules that naturally accompany it. Typically, apolypeptide is substantially pure when it is at least 60%, by weight,free from the proteins and naturally occurring organic molecules withwhich it is naturally associated. For example, the polypeptide can be apolypeptide that is at least 75%, 90%, or 99%, by weight, pure. Asubstantially pure polypeptide can be obtained, for example, byextraction from a natural source, by expression of a recombinant nucleicacid molecule encoding a polypeptide, or by chemical synthesis. Puritycan be measured by any appropriate method, e.g., by columnchromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

A polypeptide is substantially free of naturally associated componentswhen it is separated from those proteins and organic molecules thataccompany it in its natural state. Thus, a protein that is chemicallysynthesized or produced in a cellular system that is different from thecell in which it is naturally produced is substantially free from itsnaturally associated components. Accordingly, substantially purepolypeptides not only include those that are derived from eukaryoticorganisms, but also those synthesized in E. coli, other prokaryotes, orin other such systems.

An antibody is said to “specifically bind” to a polypeptide or fragmentif it recognizes and binds to the polypeptide (e.g., CM3 or 0.19), butdoes not substantially recognize and bind to other molecules (e.g.,non-CM3- or non-0.19-related polypeptides) in a sample, e.g., abiological sample that includes the polypeptide.

By “neutralization” and “neutralizing” is meant partial or completeattenuation of the biological effects of an AAI (e.g., CM3 and/or 0.19)(e.g., a negative gastrointestinal reaction). Such partial or completeattenuation of the biological effects of AAI results from modification,interruption, and/or abrogation of AAI stimulation of the innate immuneresponse (e.g., as exhibited in celiac disease). As one of skill in theart understands, there exist multiple modes of determining whether anagent, for example an antibody is to be classified as neutralizing.Neutralizing antibodies would, for example, block stimulation ofmonocytes or dendridic cells by AAI (e.g., CM3 and/or 0.19) in theassays described herein (e.g., by decreasing secretion of IL-8 by 70%,80%, 90%, 95%, 99%, or greater compared to control). Neutralizingantibodies can also block AAI (e.g., CM3 and/or 0.19) binding to TLR4(e.g., by decreasing binding by 70%, 80%, 90%, 95%, 99%, or greatercompared to control).

By “sample” is meant a tissue biopsy, amniotic fluid, cell, blood,serum, urine, stool, or other specimen obtained from a patient or a testsubject. For example, ELISA and other immunoassays can be used tomeasure levels of AAI (e.g., CM3 and 0.19); and PCR or RT/PCR can beused to measure the level of AAI (e.g., CM3 and 0.19) nucleic acidmolecules.

By “negative gastrointestinal reaction” is meant an innate immuneresponse triggered by AAI (e.g., CM3 and/or 0.19) that results inundesirable symptoms (e.g., those associated with celiac disease).

By “potency” is meant the degree to which a substance induces an innateimmune response against AAI protein (e.g., CM3 and/or 0.19 protein).

By “treating” is meant administering a pharmaceutical composition forprophylactic and/or therapeutic purposes or administering treatment to asubject already suffering from a disease to improve the subject'scondition or to a subject who is at risk of developing a disease. As itpertains to gastrointestinal disorders, treating can include improvingor ameliorating the symptoms of an undesired immune response triggeredby AAI (e.g., CM3 and/or 0.19), and prophylactic treatment can includepreventing the progression of a mild gastrointestinal disorder to a moreserious form. Treating may also mean to prevent the onset of an negativegastrointestinal reaction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D are a series of graphs showing IL-8, TNF-α, and MCP-1release in cells treated with the indicated compounds. The experimentsof FIGS. 1A and 1C were conducted in monocytic THP-1 cells and theexperiments of FIGS. 1B and 1D were conducted in monocytic U937 cells.

FIG. 1E is a graph showing expression of IL-8 in HT29 (intestinalepithelial), U937, and THP-1 cells treated with alpha-gliadin peptidep31-43 and a scrambled control peptide.

FIG. 1F is a graph showing expression of IL-8 in THP-1 cells treatedwith the indicated compounds and treated and untreated with proteinaseK.

FIG. 2A is a graph showing IL-8 expression in monocyte derived dendriticcells of healthy control patients or of celiac disease patients on theindicated diet treated with the indicated compound.

FIG. 2B is a graph showing IL-8 expression of monocyte derived dendriticcells from a healthy control in response to various doses ofpepsin/trypsin digested (PT) gliadin. Stimulation with LPS and PT zeinserved as positive and negative controls, respectively.

FIG. 2C is a series of graphs showing a flow cytometric analysis ofdendritic cells of healthy controls stimulated with PT gliadin.Upregulation of dendritic cell surface maturation markers can beobserved in the grey filled and the dotted histogram that represent PTgliadin and LPS stimulation, respectively, whereas PT zein stimulation(non filled) overlaps with the non stimulated control.

FIGS. 3A and 3B are graphs showing KC (rodent IL-8) and TNF-α expressionin peritoneal macrophages isolated from TLR4 deficient C3H/HeJ micecompared to C3H/HOuJ wildtype mice stimulated with LPS or PT gliadin.The TLR2 agonist Pam3CSK4 served as positive control.

FIGS. 3C and 3D are graphs showing expression of IL-8 secretion upon PTgliadin and LPS stimulation in 293 cells transfected with theTLR4-MD2-CD14 complex (3D) and in non transfected cells (3C). LPS andPMA served as positive, the TLR2 agonist Pam3CSK4 as negative controls.

FIG. 3E is a graph showing IL-8 secretion in monocyte derived dendridiccells stimulated with PT gliadin and LPS with peritoneal macrophagespreincubated with anti-TLR4 and anti-CD14 antibodies. TLR2 agonistPam3CSK4 and TLR3 agonist Poly I:C served as positive controls.

FIG. 4A is a graph showing TNF-alpha secretion in peritoneal macrophagesisolated from MyD88−/− mice compared to C57BL/6 wildtype mice upon PTgliadin stimulation. LPS served as positive control for the MyD88knockdown, TLR3 agonist Poly I:C served as cell viability control.

FIG. 4B is a graph showing RANTES secretion in peritoneal macrophagesisolated from C57BL/6J mice upon LPS, Poly I:C and PT gliadinstimulation.

FIG. 4C is a graph showing stimulation of THP-1 cells with α-, γ-, ω1.2and ω5-gliadin fractions isolated from the pure wheat strain ‘Rektor’.Co-incubation of α- and γ-gliadin with regular PT gliadin from Sigmaserved as cell viability control.

FIGS. 4D and 4E are graphs showing IL-8 secretion in TLR4 transfected(4D) and untransfected (4E) HEK-293 cells treated with ω-gliadins.

FIG. 4F is a graph showing IL-8 secretion in TLR4 transfected HEK-293cells treated with synthetic overlapping 20 mers of co5-gliadin. Forillustration purposes, nine fractions each were pooled in thestimulation experiments, as well as all 43 fractions. LPS served aspositive and Pam3CSK4 or PT zein as negative controls.

FIGS. 5A and 5B are graphs showing KC expression by monocyte deriveddendritic cells stimulated with water-soluble (ws) gliadin (5A) or alphaamylase trypsin inhibitor (ATI, 5B).

FIG. 5C is a graph showing IL-8 secretion in dendritic cells treatedwith a proteinase K digestion of ATI and LPS.

FIG. 5D is a graph showing KC secretion in peritoneal macrophagesisolated from MyD88−/− mice compared to C57BL/6J wildtype mice upon ATIor ws gliadin stimulation.

FIGS. 5E and 5F are graphs showing IL-8 secretion upon PT gliadin, ATI,or LPS stimulation in 293 cells transfected with the TLR4-MD2-CD14complex (5E) and in non transfected cells (5F). LPS and water-solublezein served as positive and negative controls, respectively.

FIG. 6A is a graph showing IL-8 secretion in monocyte derived dendriticcells stimulated with ATI and LPS and preincubated with anti-TLR4 oranti-CD14 antibodies. TLR2 agonist Pam3CSK4 served as positive control.

FIGS. 6B and 6C are graphs showing serum cytokine levels in C57BL/6J,MyD88−/− (6B), and Rag1−/− mice (6C) (n=4 animals per group) havingundergone intraperitoneal injection of LPS (1 μg mouse), ws gliadin (500μg/g mouse), or ws zein (500 μg/g mouse). Serum was taken 2 hrs afterinjection and serum cytokine levels were measured by ELISA.

FIGS. 6D, 6E, and 6F are graphs showing cytokine mRNA expression levelsin mice gavaged with LPS (20 μg/g mouse weight), ws gliadin (2 mg/gmouse weight), ATI (0.075 mg/g mouse weight), ws zein (2 mg/g mouseweight), or PBS. 4 hrs after gavage the C57BL/6J, C3H/HeJ, and C3H/HouJmice were euthanized and duodenal samples were snap frozen in liquidnitrogen. Duodenal cytokine mRNA levels were measured by quantitative RTPCR. (n=3 animals per group).

FIG. 7A is a western blot showing the major ATI variant CM3 wasexpressed in eukaryotic 293 cells and purified as Flag-tagged molecule,yielding the expected molecular weights of 16 kD.

FIG. 7B is a graph showing IL-8 secretion in human dendritic cells uponstimulation with ATI and the recombinant CM3 variant.

FIG. 7C is a western blot showing expression of NF-κB and thealternative (IRF3) pathways in CM3 and TLR4-transfected HEK cells.

FIG. 7D is a photomicrograph showing apoptosis in TLR4- andCM3-transfected and untransfected HEK cells.

FIGS. 8A-8C are supplementary graphs showing expression of IL-8 (8A),TNF-α (8B), and RANTES (8C) in gliadin stimulated human monocyte derivedDC from healthy controls.

FIG. 9A is a western blot showing CM3 and 0.19 expression in HEK-293cells and purification as Flag-tagged molecules. Western analysisyielded the expected molecular weights of 15 and 17 kDa, respectively,after 15% SDS-PAGE and Western blotting with Flag antibody. Noexpression in mock (vector only) transfected cells. 0.19 was notsecreted into media, but could be recovered in SDS/DTT buffer (Cell),deoxycholate and Triton X-100 buffer (Sup), or the remaining cellpellet.

FIG. 9B is a Coomassie blue stain (left panel) or western blot usingFlag antibody (right panel) showing purity of CM3 and 0.19 ATI afteraffinity purification on Flag-agarose (15% SDS-PAGE).

FIG. 9C is a graph showing IL-8 secretion by TLR4-CD14-MD2 expressingHEK-293 cells treated with recombinant ATI. Cells were left untreated,or stimulated with affinity purified detergent extracts frommock-transfected cells, from recombinant CM3, 0.19, or with ATI isolatedfrom wheat (all 5 μg/ml). Controls were Flag peptide (5 μg/ml),PT-gliadin (100 μg/ml), and LPS (10 ng/ml). Representative experimentperformed in triplicate.

FIG. 9D is a western blot showing expression of CM3 and 0.19 inTLR4-CD14-MD2 expressing HEK-293 cells (HEK-TLR4) induces downstreamsignaling of the canonical (p-NF-κB/p65) and the alternative (p-IRF3)pathways. Western blots of whole cell lysates (50 μg protein).

FIGS. 10A and 10B are a sequence alignments. FIG. 10A shows an alignmentbetween CM3 and 0.19. FIG. 10B shows an alignment between CM3 or 0.19and other ATIs from wheat and barley (CMa, CMb, CMd) (SEQ ID NOs, inorder listed are SEQ ID NOs:: 3-5, 1, 6-10, 2, and 11). The sequencealignment was prepared using the ClustalW program (2.0.12 version). Thehighly conserved cysteine residues are boxed. (*), (:), and (.) denoteidentity, high homology, and low homology, of amino acid residues,respectively. Similarities are much higher among individual ATIs.

DETAILED DESCRIPTION OF THE INVENTION

In general, the invention features the treatment of gastrointestinaldisorders associated with an innate immune response triggered by alphaamylase inhibitor CM3, alpha amylase inhibitor 0.19 (0.19), CM1, CM2,CMa, CMd, CM16, CMb, CMX1/CMX3, CMX2, alpha Amylase Inhibitor 0.53(0.53) (collectively termed AAI), and other structurally andfunctionally related molecules. To this end, the invention providespharmaceutical compositions including neutralizing antibodies to alphaamylase inhibitor CM3, alpha amylase inhibitor 0.19 (0.19), CM1, CM2,CMa, CMd, CM16, CMb, CMX1/CMX3, CMX2, alpha Amylase Inhibitor 0.53(0.53), and other structurally and functionally related molecules, foodproducts containing reduced levels of AAI proteins, assays foridentifying AAI protein content in food products, and assays fordiagnosing subjects with a disorder related to AAI-triggered innateimmune responses. The invention also features methods of treatinggastrointestinal disorders associated with an innate immune response tothese molecules with preferably, but not exclusively orally active TLR4inhibitors.

CM3

We discovered that CM3 and/or 0.19 (among other AAIs) contributes to theactivation of innate immunity in patients with celiac disease. Anexemplary wheat CM3 protein has the sequence of

(SEQ ID NO: 1) MACKSSCSLLLLAAVLLSVLAAASASGSCVPGVAFRTNLLPHCRDYVLQQTCGTFTPGSLPEWMTSASIYSPGKPYLAKLYCCQELAEISQQCRCEALRYFIALPVPSQPVDPRSGNVGESGLIDLPGCPREMQWDFVRLLVAPGQCNLATIHNVRYCPAVEQPLWIDYKDDDDK.An exemplary wheat 0.19 protein has the sequence of:

(SEQ ID NO: 2) SGPWMCYPGQAFQVPALPACRPLLRLQCNGSQVPEAVLRDCCQQLAHISEWCRCGALYSMLDSMYKEHGAQEGQAGTGAFPRCRREVVKLTAASITAVCRLPIVVDASGDGAYVCKDVAAYPD

The invention provides methods for the treatment of negativegastrointestinal reactions to other cereal-based (e.g., barley, rye,oats, corn, and rice) CM3 or 0.19 proteins, or related molecules, e.g.,those set forth in Tables 1 and 2.

TABLE 1 Accession Description P17314.1 Alpha-amylase/trypsin inhibitorCM3; P11643.2 Alpha-amylase/trypsin inhibitor CMd; [Hordeum vulgare]CAA49536.1 CMd subunit of tetrameric alpha-amylase inhibitor [Hordeumvulgare] AAB63440.1 CMd3 protein [Hordeum vulgare] CAA31585.1 CMdpreprotein (AA −14 to 146) [Hordeum vulgare subsp. vulgare] 1208404Btrypsin/amylase inhibitor pUP38 P16159.1 Alpha-amylase/trypsin inhibitorCM16; [Triticum aestivum] P32936.2 Alpha-amylase/trypsin inhibitor CMb;[Hordeum vulgare subsp. vulgare]

TABLE 2 Accession Description HSSA Alpha-amylase/trypsin inhibitor 0.19[Triticum aestivum] HSSB Alpha-amylase/trypsin inhibitor 0.19 [Triticumaestivum] HSSC Alpha-amylase/trypsin inhibitor 0.19 [Triticum aestivum]HSSD Alpha-amylase/trypsin inhibitor 0.19 [Triticum aestivum] PO1085.1Alpha-amylase/trypsin inhibitor 0.19 [Aegilops tauschii] BAA20139.1Alpha-amylase/trypsin inhibitor 0.19 [Aegilops tauschii]

A sequence comparison with a wide spectrum of other ATIs from wheat andbarley (those sequences that are published in common sequence databases)show again largely conserved cysteine residues and neighbouring aminoacid residues (FIG. 10B), indicating that these will also bind to andactivate TLR4. Additional variants of CM3 and 0.19 and related moleculesare known in the art and can be identified through standard means.

TLR4 Inhibitors

TLR4 inhibitors for pharmaceutical use are known in the art. Suchinhibitors include synthetic gluco-disaccharides such as RSCL-0409(Kalluri et al. FEBS J. 2010 April; 277(7):1639-52); TLR4 (MD2-TLR4)blocking antibodies (Ungaro et al. Am J Physiol Gastrointest LiverPhysiol. 2009 June; 296(6):G1167-79); phosphatidyl-ethanolamine (Lee etal. Mol Cells. 2009 Feb. 28; 27(2):251-5); peptide antagonists (Slivkaet al. Chembiochem. 2009 Mar. 2; 10(4):645-9); eritoram tetrasodium,E5564 (Rossignol et al. Innate Immun. 2008 December; 14(6):383-94; Kimet al. Cell. 2007 Sep. 7; 130(5):906-17; and Shimamoto et al.Circulation. 2006 Jul. 4; 114(1 Suppl):I270-4); tetra- orpenta-acetylated lipid A (Zhang et al. Org Biomol Chem. 2008 Sep. 21;6(18):3371-81; Coats et al. J Immunol. 2005 Oct. 1; 175(7):4490-8);blocking LPS variants such as LPS from cyanobacteria, Rhodobactercapsulates, Porphyromonas gingivalis and Capnocytophagatochracea orHelicobacter pylori (Ianaro et al. Mini Rev Med Chem. 2009 Mar;9(3):306-17; Jemmett et al. Infect Immun. 2008 July; 76(7):3156-63; andMacagno A, et al. J Exp Med. 2006 Jun. 12; 203(6):1481-92), BartonellaQuintana (Popa et al. Infect Immun 2007 October; 75(10):4831-7),Treponema (Lee et al. Microbiology. 2006 February; 152(Pt 2):535-46), orlipid IVa (Saitoh et al. Int Immunol. 2004 July; 16(7):961-9); E5531(Bryant et al. Vet Immunol Immunopathol. 2007 Apr. 15; 116(3-4):182-9);soluble MD-2/TLR4 complex (Mitsuzawa et al. J Immunol. 2006 Dec. 1;177(11):8133-9); including indirect negative modulators of TLR4(MD2-TLR4), such as vitamin D (Sadeghi et al. Eur J Immunol. 2006February; 36(2):361-70), testosterone derivatives (Norataet al. J ClinEndocrinol Metab. 2006 February; 91(2):546-54), CRX526 (Ianaro et al.),or inhibitors of the nicotinic acetylcholine receptor (Hamano et al.Shock. 2006 October; 26(4):358-64). Each of the above references isincorporated by reference in its entirety.

Any of the agents employed according to the present invention may becontained in any appropriate amount in any suitable carrier substance,and is generally present in an amount of 1-95% by weight of the totalweight of the composition. The composition may be provided in a dosageform that is suitable for the oral, parenteral (e.g., intravenously,intramuscularly), rectal, cutaneous, nasal, vaginal, inhalant, skin(patch), or ocular administration route. Thus, the composition may be inthe form of, e.g., tablets, capsules, pills, powders, granulates,suspensions, emulsions, solutions, gels including hydrogels, pastes,ointments, creams, plasters, drenches, osmotic delivery devices,suppositories, enemas, injectables, implants, sprays, or aerosols. Thepharmaceutical compositions may be formulated according to conventionalpharmaceutical practice (see, e.g., Remington: The Science and Practiceof Pharmacy, 20th edition, 2000, ed. A. R. Gennaro, Lippincott Williams& Wilkins, Philadelphia, and Encyclopedia of Pharmaceutical Technology,eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).

Indications

The invention provides methods for the treatment of AAI (e.g., CM3-and/or 0.19)-related gastrointestinal disorders by administering asubject (e.g., a human) in need thereof an effective amount of acomposition that includes an antibody against an AAI protein (e.g.,CM3), or a different antibodies against the major AAI variants incereals. Such disorders are related to the induction of an innate immuneresponse against AAI proteins (e.g., against CM3 and/or 0.19) after theconsumption of an AAI containing food product. These disorders includeceliac disease, ulcerative colitis, Crohn's disease, irritable bowelsyndrome, gastrointestinal hypersensitivity to wheat, as well as otherinflammatory disease of the GI tract and related immune and autoimmunedisorders.

Anti-AAI Antibodies

The invention provides therapeutic antibodies (e.g., neutralizingantibodies) and diagnostic antibodies against AAI, e.g., against CM3and/or 0.19.

The antibodies (e.g., monoclonal, polyclonal, poly-specific, ormono-specific antibodies) against AAI (e.g., against CM3 and/or 0.19)can be used for diagnostic, research, or therapeutic purposes. Numerousmethods for making antibodies are known in the art and can be used inthe invention to make such antibodies. In one example, a coding sequencefor an AAI (e.g., a CM3 and/or 0.19) peptide or polypeptide is expressedas a C-terminal fusion with glutathione S-transferase (GST) (Smith etal., Gene 67:31, 1988). The fusion protein is purified onglutathione-Sepharose beads, eluted with glutathione, cleaved withthrombin (at an engineered cleavage site), and purified for immunizationof rabbits. Primary immunizations are carried out with Freund's completeadjuvant and subsequent immunizations with Freund's incomplete adjuvant.Antibody titers are monitored by Western blot and immunoprecipitationanalyses using the thrombin-cleaved protein fragment of the GST fusionprotein. Immune sera are affinity purified using CNBr-Sepharose-coupledprotein. Antiserum specificity can be determined using a panel ofunrelated GST proteins.

As an alternate or adjunct immunogen to GST fusion proteins, peptidescorresponding to relatively unique immunogenic regions of a polypeptideof the invention can be generated and coupled to keyhole limpethemocyanin (KLH) through an introduced C-terminal lysine. Antiserum toeach of these peptides is similarly affinity purified on peptidesconjugated to BSA, and specificity is tested by ELISA or Western blotanalysis using peptide conjugates, or by Western blot orimmunoprecipitation using the polypeptide expressed as a GST fusionprotein. Alternatively, monoclonal antibodies that specifically bind anAAI (e.g., CM3 or 0.19) can be prepared using standard hybridomatechnology (see, e.g., Kohler et al., Nature 256:495, 1975; Kohler etal., Eur. J. Immunol. 6:511, 1976; Kohler et al., Eur. J. Immunol.6:292, 1976; Hammerling et al., Monoclonal Antibodies and T CellHybridomas, Elsevier, N.Y., 1981). Once produced, monoclonal antibodiescan also be tested for specific recognition by Western blot orimmunoprecipitation analysis. Antibodies that specifically recognize anAAI protein (e.g., CM3 or 0.19) can be used, for example, inimmunoassays or as therapies. Alternatively monoclonal antibodies can beprepared using the polypeptide of the invention described above and aphage display library (Vaughan et al., Nature Biotech. 14:309, 1996).

Antibodies of the invention can be produced using fragments of the AAI(e.g., a CM3 and/or 0.19) polypeptide that lie outside generallyconserved regions and appear likely to be antigenic by criteria such ashigh frequency of charged residues. In one specific example, suchfragments are generated by standard techniques of PCR and cloned intothe pGEX expression vector. Fusion proteins are expressed in E. coli andpurified using a glutathione agarose affinity matrix. To minimizepotential problems of low affinity or specificity of antisera, two orthree such fusions are generated for each protein, and each fusion isinjected into at least two rabbits. Antisera are raised by injections ina series, and can include, for example, at least three boosterinjections.

In addition to intact monoclonal and polyclonal anti-AAI (e.g., anti-CM3and/or 0.19) antibodies, the invention also includes various geneticallyengineered antibodies, humanized antibodies, chimeric antibodies, andantibody fragments, including F(ab′)2, Fab′, Fab, Fv, and sFv fragments.Truncated versions of monoclonal antibodies, for example, can beproduced by recombinant methods in which plasmids are generated thatexpress the desired monoclonal antibody fragment(s) in a suitable host.Antibodies can be humanized by methods known in the art, e.g.,monoclonal antibodies with a desired binding specificity can becommercially humanized (Scotgene, Scotland; Oxford Molecular, Palo Alto,Calif.). Fully human antibodies, such as those expressed in transgenicanimals, are also included in the invention (Green et al., NatureGenetics 7:13-21, 1994).

Examples of approaches that can be used to generate antibodies of theinvention include the following. Ladner (U.S. Pat. Nos. 4,946,778 and4,704,692) describes methods for preparing single polypeptide chainantibodies. Ward et al., Nature 341:544-546, 1989, describes thepreparation of heavy chain variable domains, which they term “singledomain antibodies,” and which have high antigen-binding affinities.McCafferty et al., Nature 348:552-554, 1990, shows that completeantibody V domains can be displayed on the surface of fd bacteriophage,that the phage bind specifically to antigen, and that rare phage (one ina million) can be isolated after affinity chromatography. Boss et al.,U.S. Pat. No. 4,816,397, describes various methods for producingimmunoglobulins, and immunologically functional fragments thereof, thatinclude at least the variable domains of the heavy and light chains in asingle host cell. Cabilly et al., U.S. Pat. No. 4,816,567, describesmethods for preparing chimeric antibodies.

Antibodies to AAI (e.g., to CM3 or 0.19) can be used, as noted above, todetect AAI, or to inhibit the biological activities of AAI. In addition,the antibodies can be coupled to compounds, such as radionuclides andliposomes, for diagnostic uses.

In order to generate polyclonal antibodies on a large scale and at a lowcost an appropriate animal species can be chosen. Polyclonal antibodiescan be isolated from the milk or colostrum of, e.g., immunized cows.Bovine colostrum contains 28 g of IgG per liter, while bovine milkcontains 1.5 g of IgG per liter (Ontsouka et al. J. Dairy Sci.86:2005-2011, 2003). Polyclonal antibodies can also be isolated from theyolk of eggs from immunized chickens (Sarker et al. J. Ped. Gastro.Nutr. 32:19-25, 2001).

Multiple adjuvants are approved for use in dairy cows. Adjuvants usefulin this invention include, but are not limited to, Emulsigen®, anoil-in-water emulsified adjuvant, Emulsigen®-D, an oil-in-wateremulsified adjuvant with DDA immunostimulant, Emulsigen®-P, anoil-in-water emulsified adjuvant with co-polymer immunostimulant,Emulsigen®-BCL, an oil-in-water emulsified adjuvant with blockco-polymer immunostimulant, Carbigen™, a carbomer base, and Polygen™, aco-polymer base. All of the listed adjuvants are commercially availablefrom MVP Laboratories in Omaha, Nebr.

Antibodies useful in this invention can be identified in severaldifferent screening assays. First, antibodies are assayed by ELISA todetermine whether they are specific for the immunizing antigen (e.g.,CM3 and/or 0.19). Using standard techniques, ELISA plates are coatedwith immunogen, the antibody is added to the plate, washed, and thepresence of bound antibody detected by using a second antibody specificfor the Ig of the species in which the antibody was generated.

A functional in vitro assay can be used to screen antibodies e.g., anneutralizing assay based on monocyte derived dendritic cells, asdescribed herein.

Direct measurements of bovine immunoglobulin in illeal fluid in humansubjects has shown that significant amounts of immunoglobulin survivetransit through the stomach and small intestine (Warny et al. Gut,44:212-217, 1999). Methods have also been described to formulate avianimmunoglobulin (IgY) for GI delivery (Kovacs-Nolan and Mine Immunol.Methods. 296: 199-209, 2005).

The invention provides a therapeutic composition comprising anti-AAI(e.g., anti-CM3 and/or anti-0.19) antibodies suitable for delivery,preferably oral delivery, to a patient in need thereof, preferably ahuman patient. The pharmaceutical composition may further comprisesuitable carriers, adjuvants and other physiologically acceptableexcipients.

The invention also features a therapeutic composition containingantibodies with specificity for both the CM3 of SEQ ID NO:1 (or proteinssubstantially identical to the protein of SEQ ID NO:1) and/or the 0.19of SEQ ID NO:2 (or proteins substantially identical to the protein ofSEQ ID NO:2) as well as additional CM3- or 0.19-related proteins (e.g.,CM3 or 0.19 analogs expressed in other species of cereals or other AAIproteins as disclosed herein). Such antibodies can be identified bytesting specificity to all of the desired CM3- or 0.19-related proteins.

Furthermore, the invention also features pharmaceutical compositionscontaining a plurality of antibodies, where different antibodies in thecomposition are specific for different analogs of AAI (e.g., CM3 and/or0.19). Additionally or alternatively, such compositions can containantibodies against both AAI (e.g., CM3 and/or 0.19), as well as gluten(e.g., gliadin and glutenin fractions, see, e.g., WO 2007/056301, whichis herein incorporated by reference in its entirety). Suchpharmaceutical compositions can be achieved by mixing more than onesource of antibodies, or, e.g., inoculating an antibody producing animalwith more than one AAI (e.g., a cow can be inoculated with variants ofCM3 and/or 0.19 with adjuvants as described above thereby creating amixture of polyclonal antibodies with differing specificities).

The oral formulations can comprise enteric coatings, so that the activeagent is delivered to the intestinal tract. Enteric formulations areoften used to protect an active ingredient from the strongly acidcontents of the stomach. Such formulations are created by coating asolid dosage form with a film of a polymer that is insoluble in acidenvironments and soluble in basic environments. Exemplary films arecellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropylmethylcellulose phthalate, and hydroxypropyl methylcellulose acetatesuccinate, methacrylate copolymers and cellulose acetate phthalate.

The pharmaceutical compositions can be administered in any orallyacceptable dosage form including, but not limited to, capsules, tablets,aqueous suspensions or solutions.

Neutralizing antibodies can be administered to a patient prior to, orconcurrently, or after the ingestion of a substance that may contain anAAI (e.g., CM3 and/or 0.19). The neutralizing antibodies of theinvention can also be administered to the patient on a regular dosingschedule (e.g., one, two, three times a day, or more). The neutralizingantibodies can also be administered at a specific time of day, e.g.,prior to sleep or upon wakening.

Detection and measurement of indicators of efficacy may be measured by anumber of available diagnostic tools, including but not limited to, forexample, by physical examination including blood tests, biopsies of thesmall intestine, pulmonary function tests, and chest X-rays; CT scan;bronchoscopy; bronchoalveolar lavage; lung biopsy and CT scan.Suppression of the innate immunity can be measured, e.g., by quantifyingthe release of cytokines at the sites of lesions. Also, both thephysician and patient can identify a reduction in symptoms of a disease.

The pharmaceutical compositions of this invention comprise any of thecompounds of the present invention, or pharmaceutically acceptablederivatives thereof, together with any pharmaceutically acceptablecarrier.

The dosage and dose rate of the compounds of this invention effective toproduce the desired effects will depend on a variety of factors, such asthe nature of the antibody, the size of the subject, the goal of thetreatment, the nature of the pathology to be treated, the specificpharmaceutical composition used, and the judgment of the treatingphysician. Dosage levels of between about 0.1 and about 1000 mg/kg bodyweight per dose, preferably between about 1 and about 500 mg/kg bodyweight per dose of the active ingredient antibody are useful. Theantibodies of the invention can also be administered at a dose rangingbetween about 1 mg/kg body weight/dose and about 200 mg/kg bodyweight/dose, and between about 5 mg/kg body weight/dose and about 50mg/kg body weight/dose.

Detection Assays

The invention features assays for detecting AAI (e.g., CM3 and/or 0.19)in food products. The assays can contain, e.g., antibodies as describedabove. Such antibodies can be antibodies specific for AAI proteins(e.g., CM3 and/or 0.19), and can have neutralizing or non-neutralizingactivities. Such antibodies can be detectably labeled to facilitatedetection. Assays for detecting proteins in a sample (e.g., a foodproduct) are known in the art (e.g., ELISA or RIA assays). Such assayscan be readily adapted to detection of AAI (e.g., CM3 and/or 0.19).

The invention also features assays for detecting antibodies against AAI(e.g., CM3 or 0.19) in a patient sample (e.g., using an ELISA assay asdescribed above). Such assays can be used, e.g., to diagnose a patientas being sensitive to AAI (e.g., sensitive to CM3 and/or 0.19, and ashaving celiac disease, ulcerative colitis, Crohn's disease, or irritablebowel syndrome). The patient sample can be any sample likely to containantibodies against AAI (e.g., a blood sample or stool sample). Thepresence or heightened levels of endogenously generated antibodiesagainst AAI can be indicative of a subject as being sensitive to AAIcontaining food products.

AAI Depleted Food Products

The invention also features food products with decreased levels of AAI(e.g., CM3 and/or 0.19). Such food products can be prepared, e.g., bydegrading or removing AAI prior to consumption. Such food products canalso be prepared from transgenic cereals (e.g., wheat) containingnucleic acid constructs encoding RNAi molecules against AAI.

Decreased levels of AAI (e.g., CM3 and/or 0.19) protein can be achieved,e.g., through disulfide reduction of AAI. This can be achieved by agentssuch dithioerithrol, or preferably by a NADP-thioredoxin system asdescribed by Farid et al. (J Agricult Food Chem 56:7146-7150, 2008) forthe improvement of digestability of soy flour. The latter and relatedmethodologies have the advantage that disulfide reduction is non-toxic.The reduced AAIs are then easily degraded by gastric and duodenalproteases, leading to their “detoxification,” i.e., inability to elicitan innate immune response. To secure complete degradation beforeingestion, the reduced AAIs can be predigested with common proteasessuch as pepsin, trypsin, or chymotrypsin, or with specialized enzymepreparations.

Decreased levels of AAI (e.g., CM3 and/or 0.19) can also be achieved byseparating AAI from the food product by, e.g., exposing the food productto antibodies specific for AAI. Such antibodies can be used, e.g., in apurification column to remove AAI from a solution containing AAI.

Methods for silencing gene expression in plants is well understood inthe art. The current invention features RNAi molecules specific for anAAI (e.g., against a nucleic acid sequence corresponding to the aminoacid sequence of SEQ ID NO:1 and/or SEQ ID NO:2). Such RNAi moleculescan be expressed from a nucleic acid construct introduced into a plantcell. Desirably, expression of the RNAi construct would result in adecrease of CM3 and/or 0.19 expression in the food product of 50%, 60%,70%, 80%, 90%, 95%, 99%, or greater.

In a specific embodiment, virus induced gene silencing (VIGS) can beused to decrease CM3 (or other AAIs) expression in, e.g., wheat or othercereals. In this embodiment, a plant virus (e.g., barley stripe mosaicvirus) is engineered to contain a sequence with substantial identity toa the genomic nucleic acid sequence corresponding to CM3 (or otherAAIs). This technology is reviewed, e.g., in Cakir et al. (Crop Sci.50:S-1-S-8, 2010), which is hereby incorporated by reference in itsentirety.

Experimental Results

To examine how far gliadin elicits innate immune responses, westimulated the human monocytic cell lines THP-1 and U937 with differentconcentrations of gliadin digested with pepsin and trypsin (PT gliadin)and measured secretion of proinflammatory cytokines in the supernatant.A digest of zein, partly homolgous storage proteins from corn that lacktoxicity for patients with celiac disease, served as negative control.In accordance with other studies, only PT gliadin but not PT zein causeda dose dependent stimulation of IL-8, TNF-α, and MCP-1 secretion in bothcell lines (FIG. 1). To rule out LPS contamination as trigger of innateresponses, gliadin, LPS, and TNF-α were digested with proteinase K,completely digesting and abrogating all peptide-related activities. Asexpected, TNF-α lost its stimulatory capacity, while LPS was still ableto induce IL-8. Importantly, PT gliadin was equally inactivated (FIG.1), indicating that the stimulatory effects of gliadin were due toprotein and not LPS contamination. Moreover, while IL-8 expression wascomparable, human monocyte-derived dendritic cells (DC) stimulated withPT gliadin expressed TNF-α and RANTES to a much lesser extent than theLPS stimulated control (FIG. 8).

It was shown previously that the a-gliadin peptide p31-43 increasesIL-15 secretion in celiac biopsies. However, this peptide did not elicitsecretion of IL-8, TNF-α, or MCP-1 in our monocytic or intestinalepithelial cell lines (HT29, Caco-2 and T84), even at 40 μg/ml (FIG. 1).This suggested the presence of other potent peptide stimulators ofinnate immune responses in PT gliadin.

We then analyzed human DC derived from peripheral blood monocytes ofceliac patients on gluten free diet (gfd, n=8), on regular diet (n=3)and healthy controls (n=10). Although there were considerableinter-individual variations, all cells strongly reacted to gliadinstimulation by IL-8 secretion. Of note, there were no significantdifferences in sensitivity towards PT gliadin between celiac patients onor off gluten free diet and healthy controls, confirming recent datathat demonstrated comparable activation of PT gliadin-stimulated DC fromcontrols and CD patients. Again, PT zein had no effect on cytokineproduction (FIGS. 2A-2D).

When exposed to PT gliadin and LPS, DC also up-regulated the cellsurface maturation markers CD25, CD80, CD83, and CD86 while zein showedno effect (FIG. 2E).

A recent study suggested that gliadin may signal via MyD88, a keyadapter molecule in the toll-like receptor (TLR)/IL-10 pathway. SinceMyD88 transmits signals from several TLRs, we studied peritonealmacrophages of C3H/HeJ mice that lack TLR4 responses due to aspontaneous point mutation in the TLR4 gene. In these mice KC (IL-8) andTNF-α secretion was reduced to baseline levels after gliadin or LPSstimulation compared to syngenic C3H/HeOuJ TLR4 competent mice. Inmacrophages from both mouse strains the specific TLR2 agonist Pam3CSK4induced equally high amounts of KC and TNF-α, verifying otherwise intactMyD88 and TLR signaling and viability of the cells (FIGS. 3A-3D).

To confirm a key role of TLR4 in the innate immune response to gliadinextract, we used HEK-293 cells (that do not express TLR4 or TLR2) thatwere transfected with the human TLR4-CD14-MD2-complex. Whilenon-transfected HEK-293 cells responded neither to gliadin nor to LPSstimulation, both stimulants induced an increase of IL-8 secretion inthe transfected cells. Specificity of the transfection was demonstratedby the absence of IL-8 induction by the TLR2 agonist Pam3CSK4 (FIG. 3).

In order to examine whether gliadin also engages TLR4 in human dendriticcells, we preincubated monocyte derived DC with anti-TLR4 and CD14blocking antibodies before adding the stimulants. This significantlyreduced IL-8 production in DC stimulated with gliadin and LPS but notwith the TLR2 and TLR3 agonists Pam3CSK4 and Poly-I:C, respectively,both in DC from healthy controls (FIG. 3E) and from celiac diseasepatients.

TLR4 is unique in its ability to mediate cellular activation via twopathways: the adapter molecule MyD88 or via interferon regulatory factor3 (IRF3) pathway. Compared to C57BL/6J wildtype mice, peritonealmacrophages from MyD88 knockout mice displayed markedly reduced KC andTNF-α secretion after gliadin and LPS stimulation, indicating a majorinvolvement of the MyD88 pathway. Since TLR3 does not use MyD88 asadapter protein for signaling, we used the TLR3 agonist Poly I:C aspositive control (FIG. 4A).

To analyze potential activation of the alternate pathway, we measuredthe secretion of RANTES in supernatants of macrophages from C57BL/6Jwildtype mice. RANTES secretion was increased, suggesting that both theMyD88 dependant and independent pathway are activated by the gliadinextract (FIG. 4B). Taken together, we can conclude that gliadin-inducedsignaling is mediated via TLR4, MyD88, and TRIF, and is CD14 dependent.

We then proceeded to further define the gliadin fractions that harboredthe stimulatory activity. Gliadins from the pure wheat strain “Rektor”were separated into their α-, γ- and ω-fractions via HPLC. The α-, γ-,ω1-2-, and ω5-gliadins, as well as whole gliadin were digested withpepsin and trypsin and first tested on THP-1 monocytic cells. Neitherthe α- nor the γ-gliadins which represent more than 90% of total gliadinharbored stimulatory activity, whereas IL-8 release was induced stronglyby PT-digested ω1-2- and ω5-gliadins (FIG. 4C). The lack of stimulationby α- or γ-gliadin was not due to toxic by-products, since addition ofLPS or whole PT gliadin fully restored stimulatory capacity (FIG. 4C).As shown for whole PT gliadin, ω1-2- and ω5-gliadin strongly inducedIL-8 secretion in TLR4 transfected HEK-293 but not in untransfectedHEK-293 cells (FIGS. 4D and 4E).

Next, we used synthetic overlapping 20 mers of the ω5 gliadin chain toidentify the TLR4-stimulating peptide sequence usingTLR4-CD14-MD2-transfected HEK cells and IL-8 secretion for a readout.Surprisingly, none of the synthetic peptides triggered IL-8 secretion(FIG. 4F). This negative finding could have been due toposttranslational modifications of the gliadin or due to a particularsecondary structure that might not have been captured by the syntheticpeptides. However, since most gliadins are not significantly modifiedposttranslationally and since their secondary structure is wellreflected even by smaller peptides, we searched for other wheat proteinsthat might have co-purified with the ω gliadins. In comassie staining,one minor protein band with a molecular mass of 15 kD clearlydistinguished the ω- from the non-reactive α- and γ-gliadin fractions.When analyzed by mass spectrometry, it could be tentativelycharacterized as wheat alpha amylase inhibitor CM3 and/or 0.19. As partof the albumin fraction, wheat alpha amylase inhibitors (AAIs) arehighly disulfide-linked and water soluble proteins. It has been shownthat they partly co-purify with gliadin and glutenin preparations.Notably, the fraction of the omega-gliadins appears to contain much ofthe low molecular weight albumins that contain AAIs. Thus awater-soluble gliadin fraction showed the same properties as the PT(omega) gliadins but with an even higher stimulatory capacity than PTgliadin (FIG. 5E) and SDS-PAGE demonstrated a more prominent band with amolecular weight of 15 kD which again was confirmed as AAI by massspectrometry.

Subsequently, we used AAI purified from wheat seed to stimulate humanmonocyte derived dendritic cells. HPLC analysis of this preparationshowed one major peak confirming its purity. Both untreated andPT-digested AAI induced increased IL-8 secretion (FIG. 5), whileproteinase K digestion abrogated its stimulatory capacity, ruling outLPS contamination (FIGS. 5D and 5E). AAI was then incubated withperitoneal macrophages from MyD88−/− and C57BL/6J wildtype mice, showinga lack of KC secretion in MyD88−/− compared to wild type cells.Moreover, AAI induced upregulation of IL-8 secretion in TLR4/MD2/CD14transfected HEK-293 as compared to untransfected cells (FIGS. 5D-5F),and incubation of human monocyte derived DC with blocking TLR4 and CD14antibodies prior to addition of ATI reduced IL-8 secretion (FIG. 6C).Overall, these results confirmed the signaling pathways previouslyidentified with crude or ω-gliadin and demonstrated that it was indeedAAI that mediated the innate immune responses.

To examine whether the in vitro responses translated intophysiologically relevant in vivo responses, we used water-solublegliadin (which was available in larger quantities) in most experimentsand AAI for select studies. When injected intraperitoneally,water-soluble gliadin lead to an increase in peripheral KC and TNF-αlevels comparable to LPS, whereas water-soluble zein did not induce aresponse. No responses were found in MyD88−/− mice, neither with LPS norwith water-soluble gliadin (FIG. 6A). A recent study implicated theadaptive immune system in modulating the innate immune response. Wetherefore injected water-soluble gliadin into T and B cell deficientRag1−/− mice. Rag1−/− mice showed cytokine levels similar to the oneselicited in C57BL/6J mice, indicating that the innate response waslargely independent of adaptive immunity (FIG. 6B).

Next we analyzed local intestinal effects of orally ingested ATI. Tothis end C57BL/6J mice were gavaged with LPS, water-soluble gliadin, orPBS, followed by measurement of transcript levels of inflammatorycytokines in the proximal duodenum. Only with water-soluble gliadin didwe detect a significant upregulation of duodenal KC, MCP-1, and IL-1β(but not TNF-α) transcripts (FIG. 6D). Of note, LPS did not increase anyof these cytokines, which is likely due to its inactivation by low pH inthe stomach or by intestinal alkaline phosphatase during intestinalpassage. When the same experiment was performed in TLR4 deficientC3H/HeJ mice and the corresponding wild type mice, an increase induodenal KC transcripts was only observed in the wild type mice. Sincewater-soluble zein did not induce cytokine transcripts, an unspecificreaction to uncommon nutritional antigens could be ruled out (FIG. 6E).

Experiments were also performed with purified alpha amylase inhibitor,in order to confirm this protein antigen as trigger of wheat-inducedinnate immune response. When we gavaged C57BL/6 mice with LPS, AAI, andwater-soluble zein, AAI was indeed able to increase transcript levels ofKC, IL-1β, and IL-6 but not TNF-α in the duodenal mucosa (FIG. 6F).While initially the observed effects appeared less significant whenpurified AAI was used instead of water-soluble gliadin, this can beexplained by the much lower AAI concentrations used in these experiments(0.075 mg of purified protein vs. 2 mg of gliadin extract per g mouseweight).

AAI CM3 and 0.19 were recombinantly expressed in eukaryotic HEK 293cells, in order to exclude bacterial contaminants, to ensure correctfolding, and to identify which of both molecules was the activecompound. The affinity purified proteins were used to stimulate monocytederived DCs. Here, CM3, and to a lesser degree 0.19, upregulated IL-8secretion, suggesting that CM3 as well as 0.19 AAI were the activecompound (FIG. 7).

CM3 and 0.19, the main ATI family members, were detected by massspectrometry and were recombinantly expressed in eukaryotic HEK-293cells to prevent bacterial contaminants (LPS) and to ensure correctprotein folding (FIGS. 9A and B). Both affinity purified ATIs stimulatedTLR4/MD2/CD14-transfected but not untransfected HEK-293 cells confirmingtheir TLR4-stimulating activity. In line with eukaryotic expression, thestimulatory activity of recombinant CM3 and 0.19 was maintained afteradditional purification using an endotoxin depletion column (FIG. 9C).In addition, overexpression of both CM3 and 0.19 in TLR4/MD2/CD14expressing HEK-293 cells strongly induced the canonical and thealternative TLR4-pathway (FIG. 9D). Sequence comparison revealed thatdespite significant differences in primary sequence, both CM3 and 0.19showed 5 stretches of highly conserved amino acid residues clusteredaround cysteins, indicative of a similar secondary structure of bothATIs (FIG. 10A). Other ATI variants (i.e., AAIs) that occur in wheat orbarley showed similar homology, suggestive of comparable biologicalactivity (FIG. 10B).

Materials

Purified cell culture tested LPS (E coli 055:B5) and in wheatalpha-amylase inhibitor (AAI) type I were purchased from Sigma-Aldrich(St Louis, Mo.), the TLR2 agonistN-Palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysine×3 HCl (Pam3CSK4) and the TLR3 agonist Polyinosine-polycytidylic acid(poly-I:C)) were obtained from Invivogen (San Diego, Calif.). Otherreagents were of the highest purity available and, if not mentionedotherwise, obtained from Sigma-Aldrich.

Isolation of Gliadin Fractions and PT-Gliadin

Unless otherwise stated gliadin purchased from Sigma-Aldrich (G3375) wasused for experiments. Gliadin from the pure wheat strain ‘Rektor’ wassubfractionated as described in the art. Briefly, α, γ, ω1.2 and ω5gliadins were obtained by HPLC purification on a Nucleosil C8 column(4.6×240 mm) at 50° C., using 0.1% trifluoroacetic acid as phase A, and99.9% acetonitrile plus 0.1% trifluoroacetic acid as phase B, and agradient from 24% B to 56% B in 30 min. Detection was at 210 nm. Purityof the fractions was confirmed by SDS PAGE, aminoterminal sequenceanalysis and their characteristic amino acid composition.

Pepsin-trypsin-digested (PT-) gliadin was generated as originallydescribed with minor modifications. Briefly, gliadin was digested withpepsin in 0.1M HCL (pH 1.8) at 37° C. for 4 hrs, followed by pHadjustment to 7.8 and digestion with trypsin at 37° C. for 4 hrs(substrate:enzyme ratio of 1:200 for both reactions). Adjustment of thepH to 4.5 resulted in a precipitate which was removed by centrifugationat 2,500 rpm, and N-tosyl-chloromethyl-ketone was added to inhibitresidual trypsin activity. The supernatant containing the PT-gliadin wasdialyzed against 10 mM ammonium carbonate, pH 7.8 (molecular weightexclusion 1000D, Spectra/Por®, Serva, Heidelberg, Germany),sterile-filtered and lyophilized. A parallel digest of zein from corn(Sigma-Aldrich) was used as negative control.

The water soluble fraction of gliadin was obtained by incubating 10 g ofgliadin in 50 ml sterile water at 37° C. for 8 hrs under continuousstirring. Insolubles were removed by centrifugation and the supernatantwas sterile filtered and lyophilized.

Synthetic Peptides

Peptide p31-43 of α-gliadin (sequence LGQQQPFPPQQPY (SEQ ID NO:3 and ascrambled control peptide (sequence GLQPFQQPQPPQY (SEQ ID NO:4)) weresynthesized by AnaSpec Inc. (San Jose, Calif.). Purity of the peptideswas >80% according to HPLC and mass spectrometry analysis. Forty-three20 mer peptides with an overlap of ten amino acids covering the 440residue omega 5 gliadin were synthesized at 60-80% purity by PrimmBiotechnology (Cambridge, Mass.).

Cell Culture and In Vitro Stimulation Experiments

THP-1, U937, HEK-293 (all from ATCC, Manassas, Va.), and HEK-293 cellsstably transfected with the TLR4-CD14-MD2 complex (Invivogen) werecultured in complete RPMI or DMEM (Cellgro, Manassas, Va.) supplementedwith penicillin/streptomycin and 10% fetal calf serum at 37° C. in a 5%CO₂ atmosphere.

For isolation of peripheral monocytes 40 ml blood was obtained from 11patients with celiac disease during their diagnostic workup (median 37years, range 18-59 years), after prior informed consent. Eight patientswere on gluten free diet and in clinical remission, three patients werenewly diagnosed and therefore on a regular gluten containing diet.Control monocytes were from the buffy coat of leukopheresis concentratesof anonymous blood donors. Whole EDTA blood or leukapheresisconcentrates were subjected to density gradient centrifugation overFicoll-Hypaque (GE Healthcare, Pittsburgh, Pa.) and CD 14-positivemonocytes purified by MACS separation according to the manufacturer'sprotocol (Miltenyi, Bergisch Gladbach, Germany). For generation ofdendritic cells, monocytes were cultered in RPMI supplemented with 10%fetal calf serum, 200 U/ml rhIL-4 and 300 U/ml rhGM-CSF (both fromPeproTech, Rocky Hill, N.J.) for 6-8 days.

Murine resident peritoneal macrophages were isolated by peritoneallavage using a 3 ml syringe and 18 G needles. Three 3 ml of sterile PBSwas injected into the peritoneal cavity and reaspirated after gentlemassage of the abdomen. For further purification MACS separation forCD11b positive cells was done according to the manufacturer's protocol(Miltenyi).

For stimulation cells were seeded on polystyrene wells at a density of1×10⁶/ml. Unless otherwise stated supernatants were harvested after a 16hr incubation with various stimulants.

Exclusion of LPS Contamination

To prove that stimulatory effects were due to protein, PT-gliadin, thewheat ATI, LPS, or TNFα were incubated with or without 20 μg/mlproteinase K (Promega, Madison, Wis.) for 4 hrs at 56° C. Afterproteinase K inactivation by boiling for 5 minsute the digests were usedfor cell stimulation.

Animals

C3H/HeJ, C3H/HeOuJ and Rag1−/− mice were obtained from The JacksonLaboratory (Bar Harbour, Me.). The MyD88−/− mice were a kind gift fromS. Akira, (Osaka University, Osaka, Japan). Congenic C57BL/6J miceserved as experimental controls and were bred under the same conditionsin the same facility. All experiments were done with mice at age 5-7weeks.

In Vivo Experiments

Mice were injected intraperitoneally with water-soluble gliadin, zein(500 μg/g body weight) or LPS (1 μg/g) in 200 μl PBS, or PBS alone(negative control). 2 hrs after injection, mice were euthanized byketamine/xylazine administration and blood was drawn by intraorbitalbleeding.

For gavage experiments mice were either raised on gluten free diet(C57BL/6J and MyD88−/− mice) or put on gluten free diet for at least twoweeks (C3H/HeJ, C3H/HeOuJ mice), and starved the night before theexperiment. Gluten free feed was obtained from Research Diets (NewBrunswick, N.J.). All stimulants were diluted in PBS. Mice wereadministered gliadin, zein (2 mg/g mouse weight), LPS (20 μg/g mouseweight), ATI (0.075 mg/g mouse weight) in 200 μl PBS. Mice wereeuthanized 4 hrs after gavage and the duodenum snap frozen in liquidnitrogen.

Cytokine/Chemokine Assays

The concentration of IL-8, TNF-α, MCP-1, RANTES, and mouse IL-8 (KC) incell culture supernatants and serum samples was determined usingvalidated ELISAs (IL-8, hTNFα: BD Pharmingen, San Jose, Calif.; MCP-1,hRANTES, mRANTES, KC: R&D Systems, (Minneapolis, Minn.; mTNFα:eBioscience, San Diego, Calif.), according to the manufacturer'sprotocols.

RNA Isolation and qRTPCR

Samples from the small intestine (0.5 cm segment, 2 cm distal to thepylorus) were collected at sacrifice and snap-frozen for furtheranalysis. Total RNA isolation was performed using Trizol reagent(Invitrogen) according to the manufacturer's instructions. Exon-exonboundary spanning primer sequences were obtained from PrimerBank(http://pga.mgh.harvard.edu/primerbank/) and sequences are listed inTable [x]. Real-time PCR was performed using LightCycler 480 SYBR GreenI mastermix (Roche, Indianapolis, Ind.) and a Roche LightCycler 480system. Mouse GAPDH served as endogenous control. PCR was set up intriplicates and threshold cycle (Ct) values of the target genes werenormalized to the endogenous control. Differential expression wascalculated according to the 2-ΔΔCT method.

Blocking Experiments

Monocyte derived dendritic cells were seeded at a concentration of1×10⁶/ml in 96 well plates. Cells were pre-incubated with blockingantibodies (polyclonal rat anti-TLR4, Invivogen, 10 μg/ml), polyclonalgoat anti-CD14, R&D, 20 μg/ml) for 3 hrs at 37° C. before stimulation.

Flow Cytometry

Human monocyte derived DC were stimulated with LPS, PT gliadin, and PTzein overnight. For flow cytometry analysis, cells were pre-incubatedwith FcR blocking reagent (Miltenyi) for 15 min at 4° C. before stainingwith monoclonal antibodies (final concentration 10 μg/ml, all fromeBioscience) for 30 min at 4° C. Cells were then washed with stainingbuffer (PBS, 1% BSA), cell viability was assessed by DAPI exclusion (0.1μg/ml, Roche) and only viable cells were analyzed by flow cytometryusing a 4 laser LSRII (BD Biosciences) and Flowjo software (Tree Star,Inc.).

Recombinant Expression of AAI CM3 and 0.19 Proteins

Recombinant flag-tagged CM3 or 0.19 were generated in a eukaryoticsystem using the expression vector pCDNA (Invitrogen) and HEK 293 cells.cDNA was optimized and synthesized by genscript (sequence numberAY436554.1) and correct orientation was checked by sequencing analysis.The expressed protein was purified from supernatant with anti-flagagarose beads in batch technique according to the manufacturer'sprotocol (Sigma).

Statistical Analysis

Differences were tested for statistical significance by the unpairedt-test. p<0.05 was considered significant. In all graphs, error barsdepict standard errors of the mean.

Recombinant Expression of ATI CM3 and 0.19 Proteins

In order to exclude bacterial contaminants, recombinant flag-tagged ATICM3 and 0.19 were generated in eukaryotic HEK-293 cells, using cDNAsoptimized to fit eukaryotic codon usage (Genscript, Pscataway, N.J.; CM3and 0.19 with NCBI GenBank sequence numbers AY436554.1 and AY729672.1,respectively). cDNAs were cloned into pUC57 and correct reading framesand orientations confirmed by sequence analysis and KpnI-ApaIrestriction. CM3 and 0.19 open reading frames were then cloned intopcDNA3.1 (+) (Invitrogen). In order to increase expression level of ATI0.19, the HindIII-KpnI fragment of the PCR product was fused with theFlag-tag at the N- instead of the C-terminus using the forward primer5′-CCCAAGCTTAGCGGACCCTGGATGTGCTAC and the reverse primer5′-CGGGGTACCCCTCAGGCGTCAGGGTAAGCGGCCAC. The CM3 and 0.19 constructs werethen subcloned into the pFLAG-CMV4 vector (Sigma-Aldrich) and thecorrect orientation was confirmed by sequencing with the N-terminalsequencing primer 5′-AATGTCGTAATAACCCCGCCCC-GTTGACGC.

Subconfluent HEK-293 cells cultured on 10 cm tissue culture dishes weretransfected with 10 μg of plasmid DNA encoding CM3 and 0.19 usinglipofectamine 2000 (Invitrogen), followed by incubation in DMEM(Mediatech) containing 1% fetal bovine serum and 100 IU penicillin/100μg/ml streptomycin for 48 h. The media were obtained by centrifugationat 4,500 rpm for 10 min at 4° C. Cells were then washed with icecold-PBS [pH 7.4], resuspended in buffer C (20 mM Tris-HCl [pH 7.4], 150mM NaCl, 50 mM NaF, 1 mM Na₃VO₄, 1 mM EDTA, 0.5% TritonX-100, 0.5%sodium deoxycholate) supplemented with EDTA-free Complete proteaseinhibitors (Roche) for 15 min on ice, and then centrifuged at 14,000 rpmfor 20 min at 4° C. Aliquots of the detergent-soluble and insolublefractions were boiled at 100° C. (the pellet in SDS sample buffer),separated on a SDS-15% polyacrylamide gel, and subjected to Western blotanalysis. Protein lysates were probed with rabbit anti-flag antibody(Sigma-Aldrich), followed by horseradish peroxidase-labeled anti-rabbitIgG (Vector Laboratories, Burlingame, Calif.). Protein bands werevisualized using enhanced chemiluminescence (Thermo Scientific,Rockford, Ill.) and X-Oat 2000A processor (Kodak).

Purification of α-Amylase Inhibitors, CM3 and 0.19

While sufficient CM3 protein was secreted into the media, 0.19 ATI hadto be isolated from the detergent-soluble fraction. Briefly, HEK-cellsexpressing 0.19 were washed with ice cold-PBS, resuspended in 50 ml ofbuffer C for 15 min on ice, and centrifuged at 14,000 rpm for 20 min at4° C. Thus, the solubilized 0.19 and soluble CM3 were bound to Flag-M2agarose (Sigma-Aldrich) pre-equilibrated with buffer C by gentle shakingfor 3 h at 4° C., washed three times with buffer C, and further washedthree times with buffer D (Buffer C without detergents). Bound ATIs wereeluted with TBS [pH 7.4] containing Flag peptide (Sigma-Aldrich). Inorder to rigorously exclude endotoxin, eluted CM3 and 0.19 were alsoapplied to an entotoxin removal column (Norgen Biotek corp., Thorold,ON, Canada). ATIs were the used at 5 μg/ml to stimulate HEK-293 cellsstably transfected with the TLR4-CD14-MD2 complex, and IL-8 secretionwas quantified after 24 h using the IL-8 ELISA as described above.

Downstream Signaling Pathways Induced by CM3 and 0.19 Overexpression

HEK-293 cells stably expressing TLR4-CD14-MD2 were induced to expressCM3 or 0.19, respectively, followed by cell lysis in buffer C. Lysateswere cleared by centrifugation at 14,000 rpm for 20 min at 4° C., and 50μg of protein run on a 15% SDS-gel and subjected to Western blotting toanalyze activation of the canonical and the alternative (p-IRF3)pathways using antibodies to phosphorylated and unphosphorylated(p-NF-κB/p65) and IRF3 (all from Cell Signaling Technology, Danvers,Mass.).

Other Embodiments

Various modifications and variations of the described methods andcompositions of the invention will be apparent to those skilled in theart without departing from the scope and spirit of the invention.Although the invention has been described in connection with specificdesired embodiments, it should be understood that the invention asclaimed should not be unduly limited to such specific embodiments.Indeed, various modifications of the described modes for carrying outthe invention that are obvious to those skilled in the fields ofmedicine, immunology, pharmacology, endocrinology, or related fields areintended to be within the scope of the invention.

All publications mentioned in this specification are herein incorporatedby reference to the same extent as if each independent publication wasspecifically and individually incorporated by reference.

What is claimed is:
 1. A pharmaceutical composition comprising anantibody against alpha Amylase Inhibitor CM3 (CM3).
 2. Thepharmaceutical composition of claim 1, wherein said antibody is apolyclonal antibody.
 3. The pharmaceutical composition of claim 1,wherein said antibody is a monoclonal antibody.
 4. The pharmaceuticalcomposition of claim 3, wherein said antibody is humanized.
 5. Thepharmaceutical composition of claim 1, wherein said antibody isformulated for oral administration.
 6. The pharmaceutical composition ofclaim 5, wherein said antibody is formulated in milk or colostrum. 7.The pharmaceutical composition of claim 6, wherein said antibody isgenerated in the milk or colostrum of a mammal.
 8. The pharmaceuticalcomposition of claim 7, wherein said mammal is a cow or a goat.
 9. Thepharmaceutical composition of claim 5, wherein said antibody isformulated to be active in the intestine.
 10. A method of treating agastrointestinal disorder comprising administering the pharmaceuticalcomposition of claim
 1. 11. The method of claim 10, wherein saidgastrointestinal disorder is selected from the group consisting ofceliac disease, ulcerative colitis, and Crohn's disease.
 12. The methodof claim 10, wherein said pharmaceutical composition is administeredimmediately prior to, concomitant with or after a meal.
 13. The methodof claim 10, wherein said pharmaceutical composition is administeredonce or twice daily. 14-31. (canceled)
 32. A pharmaceutical compositioncomprising an antibody against a protein selected from the groupconsisting of Alpha Amylase Inhibitor 0.19 (0.19), CM1, CM2, CMa, CMd,CM16, CMb, CMX1/CMX3, CMX2, alpha Amylase Inhibitor 0.53 (0.53), andstructurally and functionally related compounds.
 33. The pharmaceuticalcomposition of claim 32, wherein said antibody is a polyclonal antibody.34. The pharmaceutical composition of claim 32, wherein said antibody isa monoclonal antibody.
 35. The pharmaceutical composition of claim 34,wherein said antibody is humanized.
 36. The pharmaceutical compositionof claim 32, wherein said antibody is formulated for oraladministration.
 37. The pharmaceutical composition of claim 36, whereinsaid antibody is formulated in milk or colostrum.
 38. The pharmaceuticalcomposition of claim 37, wherein said antibody is generated in the milkor colostrum of a mammal.
 39. The pharmaceutical composition of claim38, wherein said mammal is a cow or a goat.
 40. The pharmaceuticalcomposition of claim 36, wherein said antibody is formulated to beactive in the intestine.
 41. A method of treating a gastrointestinaldisorder comprising administering the pharmaceutical composition ofclaim
 32. 42. The method of claim 41, wherein said gastrointestinaldisorder is selected from the group consisting of celiac disease,ulcerative colitis, and Crohn's disease.
 43. The method of claim 42,wherein said pharmaceutical composition is administered immediatelyprior to a meal.
 44. The method of claim 43, wherein said pharmaceuticalcomposition is administered once or twice daily. 45-66. (canceled) 67.The method of claim 3, wherein said antibody is isolated from ahybridoma.
 68. The method of claim 34, wherein said antibody is isolatedform a hybridoma.